Wild-type Molecules Research Articles

Quantitative evaluation of oncogenic KRAS signaling and therapeutic effects of molecular-targeted drugs in living cells by single-molecule imaging.

76 Background: Oncogenic KRAS mutations pose challenges due to their constitutive activation and resistance to drugs in colorectal cancer. However, several aspects remain unresolved. Single-molecule imaging has revealed distinct behaviors of KRAS. Inactive KRAS wild-type (WT) molecules exhibit continuous lateral diffusion on the plasma membrane, while activated or oncogenic KRAS molecules display slower diffusion and transient trapping due to interactions with signaling molecules. These observations suggest that the overall signal intensity of KRAS within a cell is finely regulated by integrating short-term pulse signals during transient trapping. Methods: We conducted single-molecule observations of KRAS (WT, G13D, and G12D/C/V) in living colon cancer cells, both with and without molecular-targeted drugs, before and after EGF stimulation. We quantitatively analyzed their diffusional behavior to evaluate KRAS signaling and the therapeutic effects of molecular-targeted drugs. Results: The temporal fraction of trapped KRAS WT was highest at 2 minutes (6.4%) and decreased to pre-activation level (3.0%) at 5 minutes after EGF stimulation. Although the temporal fractions of trapped KRAS mutants were higher than those of KRAS WT both before stimulation (5%) and after stimulation (10%), their diffusional behaviors were mutation-specific. The temporal fraction of trapped KRAS G13D increased sharply, and those of KRAS G12D and G12C increased at a moderate rate, while that of KRAS G12V did not elevate after EGF stimulation. Cetuximab treatment significantly reduced the temporal fraction of trapped KRAS WT after stimulation, but only moderately decreased those of KRAS mutants. Notably, transient trappings of KRAS G12D and G12C were abrogated by KRAS inhibitors, and the combined therapy of KRAS inhibitors and cetuximab demonstrated additive effects. Conclusions: Our findings indicate that the mutation-specific diffusional behavior of oncogenic KRAS can be quantitatively assessed by single-molecule imaging. This method serves as a powerful tool to uncover the mechanisms underlying oncogenic KRAS signaling and evaluate the therapeutic efficacy of molecular-targeted drugs in living cells.

Direct observation of prion-like propagation of protein misfolding templated by pathogenic mutants.

Many neurodegenerative diseases feature misfolded proteins that propagate via templated conversion of natively folded molecules. However, crucial questions about how such prion-like conversion occurs and what drives it remain unsolved, partly because technical challenges have prevented direct observation of conversion for any protein. We observed prion-like conversion in single molecules of superoxide dismutase-1 (SOD1), whose misfolding is linked to amyotrophic lateral sclerosis. Tethering pathogenic misfolded SOD1 mutants to wild-type molecules held in optical tweezers, we found that the mutants vastly increased misfolding of the wild-type molecule, inducing multiple misfolded isoforms. Crucially, the pattern of misfolding was the same in the mutant and converted wild-type domains and varied when the misfolded mutant was changed, reflecting the templating effect expected for prion-like conversion. Ensemble measurements showed decreased enzymatic activity in tethered heterodimers as conversion progressed, mirroring the single-molecule results. Antibodies sensitive to disease-specific epitopes bound to the converted protein, implying that conversion produced disease-relevant misfolded conformers.

MECP2 directly interacts with RNA polymerase II to modulate transcription in human neurons

Mutations in the methyl-DNA-binding protein MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT). How MECP2 contributes to transcriptional regulation in normal and disease states is unresolved; it has been reported to be an activator and a repressor. We describe here the first integrated CUT&Tag, transcriptome, and proteome analyses using human neurons with wild-type (WT) and mutant MECP2 molecules. MECP2 occupies CpG-rich promoter-proximal regions in over four thousand genes in human neurons, including a plethora of autism risk genes, together with RNA polymerase II (RNA Pol II). MECP2 directly interacts with RNA Pol II, and genes occupied by both proteins showed reduced expression in neurons with MECP2 patient mutations. We conclude that MECP2 acts as a positive cofactor for RNA Pol II gene expression at many neuronal genes that harbor CpG islands in promoter-proximal regions and that RTT is due, in part, to the loss of gene activity of these genes in neurons.

In-Silico analysis of the structural and functional impact of SNPs in adhesion molecules associated with cardiovascular disease and the effect of potential phytochemicals on these molecules.

Adhesion molecules are one of the most important proteins playing a major role in the regulationof cell migration, apoptosis, and survival. While adhesion molecules play a pivotal role inpreserving cellular matrix and tissue integrity, these molecules are also involved in thepathogenesis of cardiovascular diseases. The cell adhesion molecules are involved inatherosclerotic plaque progression, deposition of fibrotic tissue, and thrombus formation. Selectins-E and -P and members of the immunoglobulin superfamily ICAM-1 and ICAM-3 are the majorgroups of the adhesion molecules involved in cardiovascular diseases.In the current study, computational analysis was performed to understand the effects of missensemutations on the structure and functions of the adhesion molecules. The computational studiescan lead to a better understanding of the role of single nucleotide polymorphic mutations on thefunctions of adhesion molecules. The single nucleotide polymorphism data of adhesion moleculeswas retrieved from gnomAD v2.1.1. Only the missense SNPs were selected, and the 3D proteinstructures of the adhesion molecules were retrieved from AlphaFold. The 3D protein structures ofthe adhesion molecules were mutated using USCF Chimera 1.14.The comparison of structural stability, structural change, and interaction with ligands wasperformed between the wild-type and mutant adhesion molecules by several bioinformatics tools.The R458W mutant of ICAM-1 was found to be destabilizing the structure of the protein.Similarly, the V88M mutant of ICAM-3 and H468Y mutant of E-selectin (SELE), along withG179R and G727Q mutants of P-selectin (SELP) have also shown structural deviations ascompared to wild-type molecules. The R367C mutant of ICAM-1, P15L mutant of ICAM-3, andT756P mutant of SELP showed a significant change in the hydrogen bonding interactions withtheir respective ligands as compared to the wild-type protein interactions with a deviation from theoriginal binding free energies. No significant changes in the hydrogen bonding interactions wereobserved in the G179R and G727Q mutants of SELP.Moreover, phytochemicals data of several medicinal plants which were effective againstcardiovascular diseases was retrieved from the Indian Medicinal Plants, Photochemistry, andTherapeutics database. The molecular docking was performed to analyze the effects ofphytochemicals on adhesion molecules. The 3,29-Dihydroxyolean-12-en-28-oic acid, aphytochemical of the Panax ginseng plant was found to have an inhibitory effect on the activesites of adhesion molecules ICAM-1, ICAM-3, SELE, and SELP. Furthermore, the results of thisstudy can be tested experimentally to confirm the effect of adhesion molecule mutations on theonset of cardiovascular diseases and the role of 3,29-Dihydroxyolean-12-en-28-oic acid as ageneral drug for the treatment of diseases caused by mutant adhesion molecules.

Single-cell analysis reveals context-dependent, cell-level selection of mtDNA.

Heteroplasmy occurs when wild-type and mutant mitochondrial DNA (mtDNA) molecules co-exist in single cells1. Heteroplasmy levels change dynamically in development, disease and ageing2,3, but it is unclear whether these shifts are caused by selection or drift, and whether they occur at the level of cells or intracellularly. Here we investigate heteroplasmy dynamics in dividing cells by combining precise mtDNA base editing (DdCBE)4 with a new method, SCI-LITE (single-cell combinatorial indexing leveraged to interrogate targeted expression), which tracks single-cell heteroplasmy with ultra-high throughput. We engineered cells to have synonymous or nonsynonymous complex I mtDNA mutations and found that cell populations in standard culture conditions purge nonsynonymous mtDNA variants, whereas synonymous variants are maintained. This suggests that selection dominates over simple drift in shaping population heteroplasmy. We simultaneously tracked single-cell mtDNA heteroplasmy and ancestry, and found that, although the population heteroplasmy shifts, the heteroplasmy of individual cell lineages remains stable, arguing that selection acts at the level of cell fitness in dividing cells. Using these insights, we show that we can force cells to accumulate high levels of truncating complex I mtDNA heteroplasmy by placing them in environments where loss of biochemical complex I activity has been reported to benefit cell fitness. We conclude that in dividing cells, a given nonsynonymous mtDNA heteroplasmy can be harmful, neutral or even beneficial to cell fitness, but that the 'sign' of the effect is wholly dependent on the environment.

Epistasis Between HLA-DRB1*16:02:01 and SLC16A11 T-C-G-T-T Reduces Odds for Type 2 Diabetes in Southwest American Indians.

We sought to identify genetic/immunologic contributors of type 2 diabetes in an indigenous American community by genotyping all study participants for both high resolution HLA-DRB1 alleles and SLC16A11 to test their risk and/or protection for T2D. These genes were selected based on independent reports that HLA-DRB1*16:02:01 is protective for T2D and that SLC16A11 associates with T2D in individuals with BMI<35kg/m2, and here test the interaction of the two loci with a more complete dataset and perform a BMI sensitivity test. We define the risk-protection haplotype of SLC16A11, T-C-G-T-T, as allele "2" of a di-allelic genetic model with three genotypes, SLC16A11*11, *12, and *22, where allele "1" is the wildtype. Both earlier findings were confirmed. Together in the same logistic model with BMI≥35, DRB1*16:02:01 remains protective, 0.73, while SLC16A11 switches from risk to protection OR = 0.57 (*22) and 0.78 (*12), respectively; an added interaction term was statistically significant (OR = 0.49 with *12). Bootstrapped (b=10,000) statistical power of interaction, 0.4801, yielded mean OR = 0.43. Sensitivity analysis demonstrated the interaction significant in BMI range 30-41. To investigate the epistasis we used the primary function of the HLA-DRB1 molecule, peptide binding and presentation, to search the entire array of 15mer peptides for both the wildtype and ancient human SLC16A11 molecules for a pattern of strong binding that was associated with risk and protection for T2D. Applying computer binding algorithms suggests the core peptide at SLC16A11 D127G, FSAFASGLL, might be key for moderating risk for T2D with potential implications for T1D.

Delivery of loaded MR1 monomer results in efficient ligand exchange to host MR1 and subsequent MR1T cell activation

MR1-restricted T cells have been implicated in microbial infections, sterile inflammation, wound healing and cancer. Similar to other antigen presentation molecules, evidence supports multiple, complementary MR1 antigen presentation pathways. To investigate ligand exchange pathways for MR1, we used MR1 monomers and tetramers loaded with 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) to deliver the antigen. Using MR1-deficient cells reconstituted with wild-type MR1 or MR1 molecules that cannot bind 5-OP-RU, we show that presentation of monomer-delivered 5-OP-RU is dependent on cellular MR1 and requires the transfer of ligand from the soluble molecule onto MR1 expressed by the antigen presenting cell. This mode of antigen delivery strengthens the evidence for post-ER ligand exchange pathways for MR1, which could represent an important avenue by which MR1 acquires antigens derived from endocytosed pathogens.

Modeling transthyretin (TTR) amyloid diseases, from monomer to amyloid fibrils.

ATTR amyloidosis is caused by deposition of large, insoluble aggregates (amyloid fibrils) of cross-β-sheet TTR protein molecules on the intercellular surfaces of tissues. The process of amyloid formation from monomeric TTR protein molecules to amyloid deposits has not been fully characterized and is therefore modeled in this paper. Two models are considered: 1) TTR monomers in the blood spontaneously fold into a β-sheet conformation, aggregate into short proto-fibrils that then circulate in the blood until they find a complementary tissue where the proto-fibrils accumulate to form the large, insoluble amyloid fibrils found in affected tissues. 2) TTR monomers in the native or β-sheet conformation circulate in the blood until they find a tissue binding site and deposit in the tissue or tissues forming amyloid deposits in situ. These models only differ on where the selection for β-sheet complementarity occurs, in the blood where wt-wt, wt-v, and v-v interactions determine selectivity, or on the tissue surface where tissue-wt and tissure-v interactions also determine selectivity. Statistical modeling in both cases thus involves selectivity in fibril aggregation and tissue binding. Because binding of protein molecules into fibrils and binding of fibrils to tissues occurs through multiple weak non-covalent bonds, strong complementarity between β-sheet molecules and between fibrils and tissues is required to explain the insolubility and tissue selectivity of ATTR amyloidosis. Observation of differing tissue selectivity and thence disease phenotypes from either pure wildtype TTR protein or a mix of wildtype and variant molecules in amyloid fibrils evidences the requirement for fibril-tissue complementarity. Understanding the process that forms fibrils and binds fibrils to tissues may lead to new possibilities for interrupting the process and preventing or curing ATTR amyloidosis.

The Impact of BRAF V600E Mutation Allele Frequency on the Histopathological Characteristics of Thyroid Cancer.

A BRAF V600E mutation in papillary thyroid cancer (PTC) has been shown to be associated with aggressive behavior. Nevertheless, not all BRAF V600E PTCs behave aggressively. Allele frequency (AF) is the number of mutated molecules divided by the total number of wild-type molecules at a specific location in the genome. The relationship between BRAF V600E AF and the histopathological features of thyroid malignancies is not well understood. We hypothesized that the BRAF V600E AF will correlate directly with aggressive histopathological behavior. The aim of this study was to examine this relationship. A retrospective chart review was performed for patients treated for BRAF V600E thyroid malignancies from 2019 to 2022 at McGill University tertiary care hospitals (n = 317). Patients with BRAF V600E-positive malignancies that included information on AF were included (n = 44). The correlation between AF and tumor histopathological features was analyzed. Out of the 44 nodules with a BRAF V600E mutation, those with aggressive features of PTC had a mean AF of 25.8%, which was significantly higher than the non-aggressive group with a mean AF of 10.25% (p = 0.020). Additionally, there was a statistically significant difference in mean AF between patients with a positive sentinel LN (29%) and those with a negative sentinel LN (17.8%) (p = 0.021). Classical PTC was present in 29.5% (13/44) of nodules, with a mean AF of 15.6%. The tall cell subtype was found in 64% (28/44) of nodules, with a mean AF of 23%. Solid and hobnail subtypes were less common in this study, and there was no statistically significant relationship between AF and histopathological subtypes (p = 0.107). Nodules smaller than 1cm had a mean AF of 13.3%, while nodules ranging from 1 2cm had a mean AF of 20.6%, and those larger than 2cm had a mean AF of 27.7%. However, no statistical difference was observed between AF and nodule size (p = 0.160). In this study, BRAF V600E mutations in conjunction with AF help to determine whether thyroid malignancies will display aggressive behavior. This pre-operative finding can help thyroid specialists to determine the extent of thyroidectomy and whether lymph node dissection is required.

Engineering high-affinity dual targeting cellular nanovesicles for optimised cancer immunotherapy.

Dual targeting to immune checkpoints has achieved a better therapeutic efficacy than single targeting due to synergistic extrication of tumour immunity. However, most dual targeting strategies are usually antibody dependent which facing drawbacks of antibodies, such as poor solid tumour penetration and unsatisfied affinity. To meet the challenges, we engineered a cell membrane displaying a fusion protein composed of SIRPα and PD-1 variants, the high-affinity consensus (HAC) of wild-type molecules, and with which prepared nanovesicles (NVs). Through disabling both SIRPα/CD47 and PD-1/PD-L1 signalling, HAC NVs significantly preserved the phagocytosis and antitumour effect of macrophages and T cells, respectively. In vivo study revealed that HAC NVs had better tumour penetration than monoclonal antibodies and higher binding affinity to CD47 and PD-L1 on tumour cells compared with the NVs expressing wild-type fusion protein. Exhilaratingly, dual-blockade of CD47 and PD-L1 with HAC NVs exhibited excellent therapeutic efficacy and biosafety. This study provided a novel biomaterial against tumoural immune escape and more importantly an attractive biomimetic technology of protein delivery for multi-targeting therapies.

In Vitro and In Vivo Chaperone Effect of (R)-2-amino-6-(1R, 2S)-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin-4(3H)-one on the C1473G Mutant Tryptophan Hydroxylase 2.

Tryptophan hydroxylase 2 (TPH2) is the key and rate-limiting enzyme of serotonin (5-HT) synthesis in the mammalian brain. The 1473G mutation in the Tph2 gene decreases TPH2 activity in the mouse brain by twofold. (R)-2-amino-6-(1R, 2S)-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin-4(3H)-one (BH4) is a pharmacological chaperone for aromatic amino acid hydroxylases. In the present study, chaperone effects of BH4 on the mutant C1473G TPH2 were investigated in vitro and in vivo. In vitro BH4 increased the thermal stability (T50 value) of mutant and wild-type TPH2 molecules. At the same time, neither chronic (twice per day for 7 days) intraperitoneal injection of 48.3 mg/kg of BH4 nor a single intraventricular administration of 60 μg of the drug altered the mutant TPH2 activity in the brain of Balb/c mice. This result indicates that although BH4 shows a chaperone effect in vitro, it is unable to increase the activity of mutant TPH2 in vivo.

Soluble wild-type ACE2 molecules inhibit newer SARS-CoV-2 variants and are a potential antiviral strategy to mitigate disease severity in COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease of 2019 (COVID-19), has caused havoc around the world. While several COVID-19 vaccines and drugs have been authorized for use, these antiviral drugs remain beyond the reach of most low- and middle-income countries. Rapid viral evolution is reducing the efficacy of vaccines and monoclonal antibodies and contributing to the deaths of some fully vaccinated persons. Others with normal immunity may have chosen not to be vaccinated and remain at risk if they contract the infection. Vaccines may not protect some immunodeficient patients from SARS-CoV-2, who are also at increased risk of chronic COVID-19 infection, a dangerous stalemate between the virus and a suboptimal immune response. Intra-host viral evolution could rapidly lead to the selection and dominance of vaccine and monoclonal antibody-resistant clades of SARS-CoV-2. There is thus an urgent need to develop new treatments for COVID-19. The NZACE2-Pātari project, comprising modified soluble angiotensin-converting enzyme 2 (ACE2) molecules, seeks to intercept and block SARS-CoV-2 infection of the respiratory mucosa. In vitro data presented here show that soluble wild-type ACE2 molecules retain the ability to effectively block the Spike (S) glycoprotein of SARS-CoV-2 variants including the ancestral Wuhan, delta (B.1.617.2) and omicron (B.1.1.529) strains. This therapeutic strategy may prove effective if implemented early during the nasal phase of the infection and may act synergistically with other antiviral drugs such as Paxlovid to further mitigate disease severity.

Mitochondrial genome study in blood of maternally inherited ALS cases

BackgroundALS is a heterogeneous disease in which different factors such as mitochondrial phenotypes act in combination with a genetic predisposition. This study addresses the question of whether homoplasmic (total mitochondrial genome of a sample is affected) and/or heteroplasmic mutations (wildtype and mutant mitochondrial DNA molecules coexist) might play a role in familial ALS. Blood was drawn from familial ALS patients with a possible maternal pattern of inheritance according to their pedigrees, which was compared to blood of ALS patients without maternal association as well as age-matched controls. In two cohorts, we analyzed the mitochondrial genome from whole blood or isolated white blood cells and platelets using a resequencing microarray (Affymetrix MitoChip v2.0) that is able to detect homoplasmic and heteroplasmic mitochondrial DNA mutations and allows the assessment of low-level heteroplasmy.ResultsWe identified an increase in homoplasmic ND5 mutations, a subunit of respiratory chain complex I, in whole blood of ALS patients that allowed maternal inheritance. This effect was more pronounced in patients with bulbar onset. Heteroplasmic mutations were significantly increased in different mitochondrial genes in platelets of patients with possible maternal inheritance. No increase of low-level heteroplasmy was found in maternal ALS patients.ConclusionOur results indicate a contribution of homoplasmic ND5 mutations to maternally associated ALS with bulbar onset. Therefore, it might be conceivable that specific maternally transmitted rather than randomly acquired mitochondrial DNA mutations might contribute to the disease process. This stands in contrast with observations from Alzheimer’s and Parkinson’s diseases showing an age-dependent accumulation of unspecific mutations in mitochondrial DNA.

Structural and Dynamic Disturbances Revealed by Molecular Dynamics Simulations Predict the Impact on Function of CCT5 Chaperonin Mutations Associated with Rare Severe Distal Neuropathies

Mutations in genes encoding molecular chaperones, for instance the genes encoding the subunits of the chaperonin CCT (chaperonin containing TCP-1, also known as TRiC), are associated with rare neurodegenerative disorders. Using a classical molecular dynamics approach, we investigated the occurrence of conformational changes and differences in physicochemical properties of the CCT5 mutations His147Arg and Leu224Val associated with a sensory and a motor distal neuropathy, respectively. The apical domain of both variants was substantially but differently affected by the mutations, although these were in other domains. The distribution of hydrogen bonds and electrostatic potentials on the surface of the mutant subunits differed from the wild-type molecule. Structural and dynamic analyses, together with our previous experimental data, suggest that genetic mutations may cause different changes in the protein-binding capacity of CCT5 variants, presumably within both hetero- and/or homo-oligomeric complexes. Further investigations are necessary to elucidate the molecular pathogenic pathways of the two variants that produce the two distinct phenotypes. The data and clinical observations by us and others indicate that CCT chaperonopathies are more frequent than currently believed and should be investigated in patients with neuropathies.

Conformational diversity in purified prions produced in vitro.

Prion diseases are caused by misfolding of either wild-type or mutant forms of the prion protein (PrP) into self-propagating, pathogenic conformers, collectively termed PrPSc. Both wild-type and mutant PrPSc molecules exhibit conformational diversity in vivo, but purified prions generated by the serial protein misfolding cyclic amplification (sPMCA) technique do not display this same diversity in vitro. This discrepancy has left a gap in our understanding of how conformational diversity arises at the molecular level in both types of prions. Here, we use continuous shaking instead of sPMCA to generate conformationally diverse purified prions in vitro. Using this approach, we show for the first time that wild type prions initially seeded by different native strains can propagate as metastable PrPSc conformers with distinguishable strain properties in purified reactions containing a single active cofactor. Propagation of these metastable PrPSc conformers requires appropriate shaking conditions, and changes in these conditions cause all the different PrPSc conformers to converge irreversibly into the same single conformer as that produced in sPMCA reactions. We also use continuous shaking to show that two mutant PrP molecules with different pathogenic point mutations (D177N and E199K) adopt distinguishable PrPSc conformations in reactions containing pure protein substrate without cofactors. Unlike wild-type prions, the conformations of mutant prions appear to be dictated by substrate sequence rather than seed conformation. Overall, our studies using purified substrates in shaking reactions show that wild-type and mutant prions use fundamentally different mechanisms to generate conformational diversity at the molecular level.

Elimination of fibrin polymer formation or crosslinking, but not fibrinogen deficiency, is protective against diet‐induced obesity and associated pathologies

BackgroundObesity predisposes individuals to metabolic syndrome, which increases the risk of cardiovascular diseases, non‐alcoholic fatty liver disease (NAFLD), and type 2 diabetes. A pathological manifestation of obesity is the activation of the coagulation system. In turn, extravascular fibrin(ogen) deposits accumulate in adipose tissues and liver. These deposits promote adiposity and downstream sequelae by driving pro‐inflammatory macrophage function through binding the leukocyte integrin receptor αMβ2. ObjectivesAn unresolved question is whether conversion of soluble fibrinogen to a crosslinked fibrin matrix is required to exacerbate obesity‐driven diseases. MethodsHere, fibrinogen-deficient/depleted mice (Fib- or treated with siRNA against fibrinogen [siFga]), mice expressing fibrinogen that cannot polymerize to fibrin (FibAEK), and mice deficient in the fibrin crosslinking transglutaminase factor XIII (FXIII–) were challenged with a high-fat diet (HFD) and compared to mice expressing a mutant form of fibrinogen lacking the αMβ2‐binding domain (Fibγ390-396A). Results and ConclusionsConsistent with prior studies, Fibγ390‐396A mice were significantly protected from increased adiposity, NAFLD, hypercholesterolemia, and diabetes while Fib‐ and siFga‐treated mice gained as much weight and developed obesity‐associated pathologies identical to wildtype mice. FibAEK and FXIII– mice displayed an intermediate phenotype with partial protection from some obesity‐associated pathologies. Results here indicate that fibrin(ogen) lacking αMβ2 binding function offers substantial protection from obesity and associated disease that is partially recapitulated by preventing fibrin polymer formation or crosslinking of the wildtype molecule, but not by reduction or complete elimination of fibrinogen. Finally, these findings support the concept that fibrin polymerization and crosslinking are required for the full implementation of fibrin‐driven inflammation in obesity.

Impacts of fast production of afucosylated antibodies and Fc mutants in ExpiCHO-S™ for enhancing FcγRIIIa binding and NK cell activation

This study has employed mammalian transient expression systems to generate afucosylated antibodies and antibody Fc mutants for rapid candidate screening in discovery and early development. While chemical treatment with the fucose analogue 2-fluoro-peracetyl-fucose during transient expression only partially produced antibodies with afucosylated N-glycans, the genetic inactivation of the FUT8 gene in ExpiCHO-S™ by CRISPR/Cas9 enabled the transient production of fully afucosylated antibodies. Human IgG1 and murine IgG2a generated by the ExpiCHOfut8KO cell line possessed a 8-to-11-fold enhanced FcγRIIIa binding activity in comparison with those produced by ExpiCHO-S™. The Fc mutant S239D/S298A/I332E produced by ExpiCHO-S™ had an approximate 2-fold higher FcγRIIIa affinity than that of the afucosylated wildtype molecule, although it displayed significantly lower thermal-stability. When the Fc mutant was produced in the ExpiCHOfut8KO cell line, the resulting afucosylated Fc mutant antibody had an additional approximate 6-fold increase in FcγRIIIa binding affinity. This synergistic effect between afucosylation and the Fc mutations was further verified by a natural killer (NK) cell activation assay. Together, these results have not only established an efficient large-scale transient CHO system for rapid production of afucosylated antibodies, but also confirmed a cooperative impact between afucosylation and Fc mutations on FcγRIIIa binding and NK cell activation.

Functional and clinical analysis of five EDA variants associated with ectodermal dysplasia but with a hard-to-predict significance.

Deficiency of ectodysplasin A1 (EDA1) due to variants of the gene EDA causes X-linked hypohidrotic ectodermal dysplasia (XLHED), a rare genetic condition characterized by abnormal development of ectodermal structures. XLHED is defined by the triad of hypotrichosis, hypo- or anhidrosis, and hypo- or anodontia. Anhidrosis may lead to life-threatening hyperthermia. A definite genetic diagnosis is, thus, important for the patients’ management and amenability to a novel prenatal treatment option. Here, we describe five familial EDA variants segregating with the disease in three families, for which different prediction tools yielded discordant results with respect to their significance. Functional properties in vitro and levels of circulating serum EDA were compared with phenotypic data on skin, hair, eyes, teeth, and sweat glands. EDA1-Gly176Val, although associated with relevant hypohidrosis, still bound to the EDA receptor (EDAR). Subjects with EDA1-Pro389LeufsX27, -Ter392GlnfsX30, -Ser125Cys, and an EDA1 splice variant (c.924+7A > G) showed complete absence of pilocarpine-induced sweating. EDA1-Pro389LeufsX27 was incapable of binding to EDAR and undetectable in serum. EDA1-Ter392GlnfsX30, produced in much lower amounts than wild-type EDA1, could still bind to EDAR, and so did EDA1-Ser125Cys that was, however, undetectable in serum. The EDA splice variant c.924+7A > G resulted experimentally in a mix of wild-type EDA1 and EDA molecules truncated in the middle of the receptor-binding domain, with reduced EDA serum concentration. Thus, in vitro assays reflected the clinical phenotype in two of these difficult cases, but underestimated it in three others. Absence of circulating EDA seems to predict the full-blown phenotype of XLHED, while residual EDA levels may also be found in anhidrotic patients. This indicates that unborn subjects carrying variants of uncertain significance could benefit from an upcoming prenatal medical treatment even if circulating EDA levels or tests in vitro suggest residual EDA1 activity.

Direct observation of prion-like propagation of misfolding in single protein molecules

Many neurodegenerative diseases feature templated conversion of natively folded proteins into misfolded isoforms. However, such prion-like propagation of misfolding has never been observed directly in any protein, hindering efforts to decipher conversion mechanisms and develop countermeasures. We observed prion-like conversion in single molecules of superoxide dismutase-1 (SOD1), which misfolds in amyotrophic lateral sclerosis (ALS), by tethering misfolded mutants associated with familial ALS to wild-type molecules held in optical tweezers.

Acoustic Array Biochip Combined with Allele-Specific PCR for Multiple Cancer Mutation Analysis in Tissue and Liquid Biopsy.

Regular screening of point mutations is of importance to cancer management and treatment selection. Although techniques like next-generation sequencing and digital polymerase chain reaction (PCR) are available, these are lacking in speed, simplicity, and cost-effectiveness. The development of alternative methods that can detect the extremely low concentrations of the target mutation in a fast and cost-effective way presents an analytical and technological challenge. Here, an approach is presented where for the first time an allele-specific PCR (AS-PCR) is combined with a newly developed high fundamental frequency quartz crystal microbalance array as biosensor for the amplification and detection, respectively, of cancer point mutations. Increased sensitivity, compared to fluorescence detection of the AS-PCR amplicons, is achieved through energy dissipation measurement of acoustically "lossy" liposomes binding to surface-anchored dsDNA targets. The method, applied to the screening of BRAF V600E and KRAS G12D mutations in spiked-in samples, was shown to be able to detect 1 mutant copy of genomic DNA in an excess of 104 wild-type molecules, that is, with a mutant allele frequency (MAF) of 0.01%. Moreover, validation of tissue and plasma samples obtained from melanoma, colorectal, and lung cancer patients showed excellent agreement with Sanger sequencing and ddPCR; remarkably, the efficiency of this AS-PCR/acoustic methodology to detect mutations in real samples was demonstrated to be below 1% MAF. The combined high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness, and compatibility with routine workflow, make this approach a promising tool for implementation in clinical oncology labs for tissue and liquid biopsy.